INFLUENCE OF FREEZING STAGES ON INFLUENZA VIRUS HAEMAGGLUTININ CONFORMATION DURING LYOPHILIZATION

Abstract

The purpose of this study was to evaluate the impact of freezing stages on influenza Haemagglutinin (HA) conformation and the influence of lyophilization cycle on properties of final lyophilized live attenuated influenza virus (LAIV) vaccine as well as their stabilities. Firstly, the effect of three freezing rates, rapid freezing (0.50oC/min), moderate freezing (1.08oC/min) and slow freezing (2.58oC/min) on HA conformational stability was investigated by far-UV, near-UV circular dichroism and fluorescence spectroscopy. The three freezing rates resulted in the loss in secondary and tertiary structures when HA powder was prepared in aqueous solution. Therefore, suitable stabilizers were added in HA formulations for better preservation during freezing cycles. Four mannitol-based formulations were tested, G6S0, G6S1, G0S1 and G0S0, where G and S were glycine and sucrose and numerals expressed as concentrations (%w/v) of each stabilizer. G6S0 formulation exhibited the smallest effect in both secondary and tertiary structures of HA after frozen at moderate freezing rate (1.08oC/min) as compared to unstabilized HA. Consequently, the moderate freezing rate of 1.08oC/min was used as the freezing stage of the final lyophilization procedure applied on LAIV vaccine formulations. Initially, lyophilization procedure resulted in acceptable LAIV products when characterized on physical appearance, reconstitution time, pH, osmolality, thermal behavior, crystallinity and infectivity titer. Only residual moisture contents were greater than specified. The stability studies were carried out under three sets of conditions: 30oC/75%RH, -20oC and 2-8oC. All formulations displayed loss in infectivity titers during the first week of storage at 30oC/75%RH. Stability determination at 2-8oC showed that only G6S0 and G6S1 products were stable throughout the 6 months study period with excellent infectivity titers similar to products stored at -20oC. In summary, glycine plays an important role in stabilizing HA conformation as well as stability of lyophilized LAIV vaccine mannitol-based products. It should be used in the future development for the manufacturing of lyophilized LAIV vaccine products.